Hepatitis C virus (HCV) infection can result in varied clinical outcomes including acute hepatitis, chronic hepatitis, cirrhosis, and hepatocellular carcinoma. More than 150,000 new infections occur in the U.S. alone with chronic infections established in >80% of those infected. Although progress has been made, a vast number of unanswered questions remain concerning HCV replication, pathogenesis and immunity. The field is rapidly reaching a bottleneck where we understand some aspects of the functions of the HCV genome RNA and its encoded proteins but have no way of experimentally testing structure/function questions in the context of authentic virus replication. Such analyses are critical for understanding each step in the virus life cycle at a level which will allow us to design protective vaccines and effective therapy for chronically infected patients. This proposal focuses on the development of full-length HCV cDNA clones capable of producing infectious RNA transcripts, a technology which is essential not only for future studies on the molecular details of HCV replication by also for defining possible links between viral genotype and clinical outcome. Relevant to this goal, a novel 3' terminal HCV sequence has been discovered which is highly conserved and likely to be essential for authentic virus replication. Areas of conservation and divergence among genotypes will be defined and clinical samples examined for possible correlations between particular 3' sequences and disease severity, interferon response, immune status, and tissue tropism. To increase our chances of recovering functional HCV clones, high fidelity reverse transcriptase/PCR will be used to construct libraries of full-length cDNA clones. From these libraries, functional clones will be identified by screening for infectivity by intrahepatic inoculation of chimpanzees or by transfecting various cell cultures with RNA transcripts. Alternatively, multiple clones from the library will be used to determine an HCV consensus sequence and for assembly of full-length clones. Functional clones will be used to produce relatively homogeneous HCV stocks for neutralization studies and to begin an examination of HCV RNA replication and particle production in cell cultures.